P3.036: Effect of human breast milk derived-exosomes in ameliorating neonatal necrotizing enterocolitis

Background: Neonatal necrotizing enterocolitis (NEC), a severe intestinal inflammatory disease, predominantly affects preterm infants and leads to high morbidity and mortality. Current therapies fail to address long-term complications like intestinal strictures and neurodevelopmental delays. Breastfeeding reduces NEC risk via bioactive components, particularly human milk-derived exosomes (HM-exos) — nanovesicles (40–160 nm) with stable bioactivity and low immunogenicity. However, the dynamic composition of breast milk (e.g., colostrum vs. mature milk) and the mechanisms underlying HM-exos’ protective effects remain unclear. This study compares colostrum-derived exosomes (C-exos) and mature milk-derived exosomes (M-exos) in repairing intestinal injury and explores their mechanisms via IGF-1/Erk signaling.

Methods: 1. In Vivo NEC Model
Animals: 40 neonatal C57BL/6 mice were divided into four groups: control (CON), NEC model (NEC), NEC+C-exos, and NEC+M-exos.
Interventions: NEC was induced via hypoxia-ischemia and cold stress; C-exos/M-exos (isolated by ultracentrifugation) were administered orally.
Analyses: Histopathology (HE staining), immunohistochemistry (ZO-1, E-cad, Lgr5, IL-6), ELISA (plasma IL-6), and qPCR (IL-6/Lgr5 mRNA).
2. In Vitro Epithelial Repair
Cell Models: IEC-6/IEC-18 cells were grouped into CON, LPS, LPS+C-exos, and LPS+M-exos.
Assays: Migration assay, scratch healing, and CCK-8 proliferation tests.
3. Mechanistic Studies
RNA Sequencing: Identified differential gene expression (e.g., Igf1) in NEC vs. CON intestinal tissues.
Pathway Analysis: Validated IGF-1 protein in HM-exos via Western blot and assessed Erk/Stat3/Akt phosphorylation.
Erk Inhibition: IEC-6 cells treated with LPS, C-exos, and Erk inhibitor (GSK143) to evaluate migration (scratch assay) and p-Erk/Erk expression.

Results: 1. In Vivo Effects
Pathology: NEC caused severe intestinal damage (highest in ileum), reduced by both C-exos and M-exos (p < 0.05), with no intergroup difference.
Barrier Function: C-exos outperformed M-exos in restoring ZO-1/E-cad expression (immunohistochemistry).
Inflammation: Both exosomes reduced IL-6 in plasma and intestinal tissue (p < 0.05).
2. In Vitro Repair
Migration/Healing: C-exos enhanced cell migration and scratch closure more effectively than M-exos (p < 0.05).
Proliferation: Both exosomes reversed LPS-induced proliferation inhibition, with no significant difference.
IGF-1 Pathway: NEC mice showed reduced Igf1 expression; HM-exos (especially C-exos) contained IGF-1 protein.
Erk Signaling: C-exos suppressed LPS-induced Erk phosphorylation (*p < 0.05*). Combining C-exos with GSK143 (Erk inhibitor) restored IEC-6 migration to baseline.

Conclusion: 1. Therapeutic Potential: C-exos and M-exos equally alleviate NEC-induced inflammation and intestinal damage, but C-exos excel in restoring barrier proteins (ZO-1/E-cad) and promoting epithelial migration.
2. Mechanistic Insight: HM-exos repair intestinal injury via IGF-1-dependent Erk pathway modulation, with C-exos delivering higher IGF-1 levels.
3. Clinical Implication: Prioritizing colostrum-derived exosomes may optimize NEC prevention and neonatal gut repair strategies.